The present invention is directed to the field of immunology and, in particular, to inactivated respiratory syncytial (RS) virus vaccines.
Human respiratory syncytial virus is the main cause of lower respiratory tract infections among infants and young children (refs. 1 to 3-a list of references appears at the end of the disclosure and each of the references in the list is incorporated herein reference thereto). Globally, 65 million infections occur every year resulting in 160,000 deaths (ref. 4). In the USA alone, 100,000 children may require hospitalization for pneumonia and bronchiolitis caused by RS virus in a single year (refs. 5, 6). Providing inpatient and ambulatory care for children with RS virus infections costs in excess of $340 million annually in the USA (ref. 7). Severe lower respiratory tract disease due to RS virus infection predominantly occurs in infants two to six months of age (ref. 8). Approximately 4,000 infants in the USA die each year from complications arising from severe respiratory tract disease caused by infection with RS virus and Parainfluenza type 3 virus (PIV-3). The World Health Organization (WHO) and the National Institute of Allergy and Infectious Disease (NIAID) vaccine advisory committees have ranked RS virus second only to HIV for vaccine development.
RS virus is a member of the Paramyxoviridae family of the pneumovirus genus (ref. 2). The two major protective antigens are the envelope fusion (F) and attachment (G) glycoproteins (ref. 9). The F protein is synthesized as a 68 kDa precursor molecule (F0) which is proteolytically cleaved into disulfide-linked F1 (48 kDa) and F2 (20 kDa) polypeptide fragments (ref. 10). The G protein (33 kDa) is heavily O-glycosylated giving rise to a glycoprotein of apparent molecular weight of 90 kDa (ref. 11). Two broad subtypes of RS virus have been defined: A and B (ref. 12) The major antigenic differences between these subtypes are found in the G glycoprotein (refs. 7, 13).
A safe and effective RS virus vaccine is not available and is urgently needed. Approaches to the development of RS virus vaccines have included inactivation of the virus with formaldehyde, isolation of cold-adapted and/or temperature-sensitive mutant viruses and isolation of the protective antigens of the virus. Clinical trial results have shown that both live attenuated and formalin-inactivated vaccines failed to adequately protect vaccinees against RS virus infection (refs. 14 to 16). Problems encountered with cold-adapted and/or temperature-sensitive RS virus mutants administered intranasally included clinical morbidity, genetic instability and overattenuation (refs. 17 to 19). A live RS virus vaccine administered subcutaneously also was not efficacious (ref. 20). Inactivated RS viral vaccines have typically been prepared using formaldehyde as the inactivating agent. Murphy et al. (ref. 21) has reported data on the immune response in infants and children immunized with formalin-inactivated RS virus. Infants (2 to 6 months of age) developed a high titre of antibodies to the F glycoprotein but had a poor response to the G protein. Older individuals (7 to 40 months of age) developed titres of F and G antibodies comparable to those in children who were infected with RS virus. However, both infants and children developed a lower level of neutralizing antibodies than did individuals of comparable age with natural RS virus infections. The unbalanced immune response, with high titres of antibodies to the main immunogenic RS virus proteins F (fusion) and G (attachment) proteins but a low neutralizing antibody titre, may be in part due to alterations of important epitopes in the F and G glycoproteins by the formalin treatment. Furthermore, some infants who received the formalin-inactivated RS virus vaccine developed a more serious lower respiratory tract disease following subsequent exposure to natural RS virus than did non-immunized individuals (refs. 15, 16). The formalin-inactivated RS virus vaccines, therefore, have been deemed unacceptable for human use.
Evidence of an aberrant immune response also was seen in cotton rats immunized with formalin-inactivated RS virus (ref. 22). Furthermore, evaluation of RS virus formalin-inactivated vaccine in cotton rats also showed that upon live virus challenge, immunized animals developed enhanced pulmonary histopathology (ref. 23).
The mechanism of disease potentiation caused by formalin-inactivated RS virus vaccine preparations remains to be defined but is a major obstacle in the development of an effective RS virus vaccine. The potentiation may be partly due to the action of formalin on the F and G glycoproteins. Additionally, a non-RS virus. specific mechanism of disease potentiation has been suggested, in which an immunological response to contaminating cellular or serum components present in the vaccine preparation could contribute, in part, to the exacerbated disease (ref. 24). Indeed, mice and cotton rats vaccinated with a lysate of HEp-2 cells and challenged with RS virus grown on HEp-2 cells developed a heightened pulmonary inflammatory response.
Furthermore, RS virus glycoproteins purified by immunoaffinity chromatography using elution at acid pH were immunogenic and protective but also induced immunopotentiation in cotton rats (refs. 22, 25).
There clearly remains a need for immunogenic preparations, including vaccines which are not only effective in conferring protection against disease caused by RS virus but also does not produce unwanted side-effects, such as immunopotentiation. There is also a need for antigens for diagnosing RSV infection and immunogens for the generation of antibodies (including monoclonal antibodies) that specifically recognize RSV proteins for use, for example, in diagnosis of disease caused by RS virus.
Art recognized approaches to the developments of RSV vaccines have been summarized in recent review articles (refs. 2, 31 to 35), none of which propose the development of an inactivated RSV vaccine.
The present invention provides a novel approach to the provision of such antigens and immunogens by inactivation of purified RS virus.
In one aspect of the present invention, there is provided a method of preparing an immunogenic composition capable of producing a respiratory syncytial (RS) virus specific immune response in a host immunized therewith, particularly a human host, which comprises a plurality of steps. The RS virus first is grown on an appropriate cell line and the virus harvested. The harvested virus is purified under non-denaturing conditions to produce a purified virus substantially free from cellular and serum components. The purified virus then is inactivated with an inactivating agent to provide a non-infectious, non-immunopotentiating and immunogenic RS virus. This RS virus then is formulated as an immunogenic composition.
The inactivating agent may be xcex2-propiolactone; a non-ionic detergent, including n-octyl-xcex1-D-glucopyranoside and n-octyl-xcex2-D-glucopyranoside; or ascorbic acid.
The purifying step which is carried out on the harvested virus preferably may be effected by microfiltration to remove cell debris, tangential flow ultrafiltration to remove serum components, particularly employing an about 100 to about 300 kDa nominal molecular weight cut-off membrane, pelleting the ultrafiltered material by ultracentrifugation to further remove serum components and subjecting the pelleted material to sucrose density gradient centrifugation. Alternatively, the retentate from tangential flow ultrafiltration may be subjected to gel filtration followed by ion-exchange chromatography to further remove serum components.
This procedure provides a novel immunogenic composition capable of producing an RS virus specific immune response in a host immunized therewith which constitutes a further aspect of the present invention. Such immunogenic composition comprises purified, inactivated RS virus which is substantially free from cellular and serum components and which is non-infectious, non-immunopotentiating, immunogenic and protective, and a carrier therefor. The immunogenic composition may be formulated as a vaccine for in vivo administration to a human host for protecting the human from a disease induced by RS virus. The carrier for the immunogenic composition may comprise an adjuvant. The immunogenic composition may be formulated as a vaccine to be administered in an injectable form, intranasally or orally.
The present invention further provides a method of immunizing a host, particularly a human host, against disease caused by RS virus, which comprises administering to the host an effective amount of the immunogenic composition provided herein. The host immunized by such procedure may be selected from infants, young children, pregnant women, women of childbearing age, elderly individuals, immunocompromised individuals and other susceptible persons.
The inactivated RS virus provided herein also may be used as a diagnostic reagent for detecting infection by RS virus. Accordingly, the present invention further includes a method of determining the presence of antibodies specifically reactive with RS virus proteins in a sample, comprising the steps of:
(a) contacting the sample with the immunogenic composition of the invention to produce complexes comprising the non-infectious, non-immunogenic and immunogenic RS virus and any antibodies present in the sample specifically reactive therewith; and
(b) determining production of the complexes.
In addition, the present invention provides a method of determining the presence of RS virus proteins in a sample, comprising the steps of:
(a) immunizing a subject with the immunogenic composition of the invention to produce antibodies specific for RS virus proteins;
(b) contacting the sample with the antibodies to produce complexes comprising any RS virus proteins present in the sample and the RS virus protein-specific antibodies; and
(c) determining production of the complexes.
The present invention further provides a diagnostic kit for determining the presence of antibodies in a sample specifically reactive with RS virus proteins, comprising:
(a) the immunogenic composition of the invention;
(b) means for contacting the non-infectious, non-immunopotentiating and non-immunogenic RS virus with the sample to produce complexes comprising the non-infectious, non-immunopotentiating and immunogenic RS virus and any said antibodies present in the sample; and
(c) means for determining production of the complexes.
Having regard to the prior art difficulty with RS virus vaccine preparations, it is surprising that the procedures described herein provide immunogenic compositions which exhibits immunogenicity and protective ability while being non-infectious and non-immunopotentiating.
In one aspect, the present invention relates to the preparation of an inactivated respiratory syncytial virus under conditions acceptable for use in human vaccines. RS virus, subtypes A or B, are grown in tissue culture in controlled fermenters on a vaccine quality cell line, which may particularly be VERO cells. Following harvesting of the virus, the virus is purified and then inactivated to produce an inactivated RS virus vaccine.
Growth of Calls
Vaccine quality cell lines, such as African green monkey kidney (VERO) cells, generally are grown on microcarrier beads (Cytodex-1). Such beads generally are swollen in a buffered solution, such as phosphate buffered saline (PBS), pH about 6.9 to about 8.2, without calcium and magnesium, for 2 to 4 hours at room temperature with gentle agitation (30 to 50 rpm). Washed beads are sterilized at 110xc2x0 C. to 130xc2x0 C. for 30 to 60 min, and conditioned in a cell culture medium, such as CMRL 1969, in spinner flasks or small (2 to 10L) or large (20 to 2000L) controlled fermenters. The vessel then is seeded (for example, 0.5xc3x97105 to 2xc3x97105 cells/mL) with vaccine quality VERO cells in a culture medium, such as CMRL 1969 supplemented with fetal bovine serum (FBS).
RB Virus Growth
Once the cells are approximately 80 to 90% confluent (3 to 5 days post-cell seeding), the culture supernatant is decanted and the cells washed once with a culture medium, such as CMRL 1969. The cells then are infected with RS virus in CMRL 1969 in the absence of FBS. After virus adsorption, the infected cells are washed in culture medium and virus growth is monitored for 5 to 7 days post-infection.
Virus Processing and Concentration
The harvested virus then is purified under non-denaturing conditions to be substantially free from cellular and serum components. Such purification may be effected in any convenient manner. In one such procedure, the RS virus supernatant is microfiltered (0.22 to 8 Mm pore size filters) to remove cell debris. The clarified viral fluid then may be concentrated by tangential flow ultrafiltration using an ultrafiltration membrane with a molecular weight cut-off between about 100 to about 300 kDa, to remove serum components.
The concentrated RS virus is subjected to further purification for utilization in the immunogenic preparations and as antigens. In one procedure, the concentrated virus may be pelleted by ultracentrifugation and the supernatant from the ultracentrifugation is discarded thereby further removing serum components. The pelleted virus is resuspended in PBS or another suitable medium. The concentrated virus then is purified by sucrose density gradient ultracentrifugation. Alternatively, the retentate from the tangential flow ultrafiltration step may be subjected to gel filtration followed by ion-exchange chromatography to further remove serum components. The resulting RS viral material may be further pelleted by ultracentrifugation. The pelleted-purified RS virus may be resuspended in PBS and stored at xe2x88x9270xc2x0 C. pending use.
Virus Inactivation
The purified RS virus next is inactivated. Such inactivation is effected using materials which provide the purified virus in a non-infectious, non-immunopotentiating and immunogenic form. The inactivating agent employed in this step generally comprises xcex2-propiolactone, ascorbic acid or a non-ionic detergent. Among the non-ionic detergents which may be employed in the inactivation step are certain glucopyranosides, including n-octyl-xcex2-D-glucopyranoside and n-octyl-xcex1-D-glucopyranoside.
Any convenient quantity of inactivating agent and any desired reaction conditions may be employed consistent with the desire to provide a non-infectious, non-immunopotentiating and immunogenic material.
As an example, RS virus may be inactivated using about a 0.1% solution of xcex2-propiolactone (BPL) for about 30 to 120 minutes or ascorbic acid for about 24 hours at about 37xc2x0 C. with constant shaking. The residual BPL may be removed from the inactivated sample by dialysis against PBS using a 10,000 to 20,000 molecular weight membrane.
Immunogenicity Studies in Cotton Rats
The purified and inactivated RS viral material provided by this procedure is immunogenic and protective while being non-infectious. This result was determined by evaluation of the immunogenicity RS viral materials in cotton rats including their capacity to protect these animals from live RS virus challenge, as reported below. Vaccine preparations provided herein elicited RS virus specific neutralizing antibodies and protected cotton rats from live virus challenge.
It is clearly apparent to one skilled in the art, that the various embodiments of the present invention have many applications in the fields of vaccination, diagnosis, treatment of diseases caused by respiratory syncytial virus and the generation of immunological reagents. A further non-limiting discussion of such uses is further presented below.
Vaccine Preparation and Use
Immunogenic compositions, suitable to be used as vaccines, may be prepared from inactivated RSV as disclosed herein. The vaccine elicits an immune response in a subject which produces anti-RSV antibodies. Should the vaccinated subject be challenged by RSV, the antibodies bind to and inactivate the virus.
Immunogenic compositions including vaccines may be prepared as injectables, as liquid solutions or emulsions. The inactivated RSV may be mixed with pharmaceutically acceptable excipients which are compatible therewith. Such excipients may include, water, saline, dextrose, glycerol, ethanol, and combinations thereof. The immunogenic compositions and vaccines may further contain auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, or adjuvants to enhance the effectiveness thereof. Methods of achieving adjuvant effect include the use of agents such as aluminum hydroxide or phosphate (alum), commonly used as 0.05 to 0.1 percent solution in phosphate buffered saline. Immunogenic compositions and vaccines may be administered parenterally, by injection subcutaneously or intramuscularly. Alternatively, the immunogenic compositions formed according to the present invention, may be formulated and delivered in a manner to evoke an immune response at mucosal surfaces. Thus, the immunogenic composition may be administered to mucosal surfaces by, for example, the nasal or oral routes. Alternatively, other modes of administration including suppositories and oral formulations may be desirable. For suppositories, binders and carriers may include, for example, polyalkalene glycols or triglycerides. Oral formulations may include normally employed incipients such as, for example, pharmaceutical grades of saccharine, cellulose and magnesium carbonate. These compositions can take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain about 1 to 95% of the inactivated RSV provided herein. The immunogenic preparations and vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective, protective and immunogenic. The quantity to be administered depends on the subject to be treated, including, for example, the capacity of the individual""s immune system to synthesize antibodies, and if needed, to produce a cell-mediated immune response. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner. However, suitable dosage ranges are readily determinable by one skilled in the art. Suitable regimes for initial administration and booster doses are also variable, but may include an initial administration followed by subsequent administrations. The dosage may also depend on the route of administration and will vary according to the size of the host.
The concentration of inactivated RS virus in an immunogenic composition according to the invention is in general about 1 to 95%. A vaccine which contains antigenic material of only one pathogen is a monovalent vaccine. Vaccines which contain antigenic material of several pathogens are combined vaccines and also belong to the present invention. Such combined vaccines contain, for example, material from various pathogens or from various strains of the same pathogen, or from combinations of various pathogens.
Immunoassays
The inactivated RSV preparations of the present invention are useful as immunogens for the generation of antibodies (including monoclonal antibodies) specifically reactive with RSV proteins as antigens in immunoassays including enzyme linked immunosorbent assays (ELISA), RIAs and other non-enzyme linked antibody binding assays or procedures known in the art for the detection of bacterial antibodies. In ELISA assays, the inactivated RSV is immobilized onto a selected surface, for example, a surface capable of binding proteins such as the wells of a polystyrene microtitor plate. After washing to remove incompletely adsorbed virus, a nonspecific protein such as a solution of bovine serum albumin (BSA) that is known to be antigenically neutral with regard to the test sample may be bound to the selected surface. This allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific bindings onto the surface.
The immobilizing surface is then contacted with a sample, such as clinical or biological materials, to be tested in a manner conducive to immune complex (antigen/antibody) formation. This may include diluting the sample with diluents, such as solutions of BSA, bovine gamma globulin (BGG) and/or phosphate buffered saline (PBS)/Tween. The sample is then allowed to incubate for from 2 to 4 hours, at temperatures such as of the order of about 25xc2x0 C. to 37xc2x0 C. Following incubation, the sample-contacted surface is washed to remove non-immunocomplexed material. The washing procedure may include washing with a solution, such as PBS/Tween or a borate buffer. Following formation of specific immunocomplexes between the test sample and the bound inactivated RSV, and subsequent washing, the occurrence, and even amount, of immunocomplex formation may be determined by subjecting the immunocomplex to a second antibody having specificity for the first antibody. If the test sample is of human origin, the second antibody is an antibody having specificity for human immunoglobulins and in general IgG. To provide detecting means, the second antibody may have an associated activity such as an enzymatic activity that will generate, for example, a colour development upon incubating with an appropriate chromogenic substrate. Quantification may then be achieved by measuring the degree of colour generation using, for example, a visible spectra spectrophotometer.